B. thuringiensis (Bt) is unique in its ability to produce, during the process of sporulation, proteinaceous, crystalline inclusions which are found to be highly toxic to several insect pests of agricultural importance. The crystal proteins of different Bt strains have a rather narrow host range and hence are used commercially as very selective biological insecticides. Numerous strains of Bt are toxic to lepidopteran and dipteran insects. Recently two subspecies (or varieties) of Bt have been reported to be pathogenic to coleopteran insects: var. tenebrionis (Krieg et al. (1983) Z. Angew. Entomol. 96:500-508) and var. san diego (Herrnstadt et al. (1986) Biotechnol. 4:305-308). Both strains produce flat, rectangular crystal inclusions and have a major crystal component of 64-68 kDa (Herrnstadt et al. Supra; Bernhard (1986) FEMS Microbiol. Lett. 33:261-265).
Toxin genes from several subspecies of Bt have been cloned and the recombinant clones were found to be toxic to lepidopteran and dipteran insect larvae. The two coleopteran-active toxin genes have also been isolated and expressed. Herrnstadt et al. supra cloned a 5.8 kb BamHI fragment of Bt var. san diego DNA. The protein expressed in E. coli was toxic to P. luteola (Elm leaf beetle) and had a molecular weight of approximately 83 kDa. This 83 kDa toxin product from the var. san diego gene was larger than the 64 kDa crystal protein isolated from Bt var. san diego cells, suggesting that the Bt var. san diego crystal protein may be synthesized as a larger precursor molecule that is processed by Bt var. san diego but not by E. coli prior to being formed into a crystal.
Sekar et al. (1987) Proc. Nat. Acad. Sci. USA 84:7036-7040; U.S. patent application Ser. No. 108,285, filed Oct. 13, 1987 isolated the crystal protein gene from Btt and determined the nucleotide sequence. This crystal protein gene was contained on a 5.9 kb BamHI fragment (pNSBF544). A subclone containing the 3 kb HindIII fragment from pNSBF544 was constructed. This HindIII fragment contains an open reading frame (ORF) that encodes a 644-amino acid polypeptide of approximately 73 kDa. Extracts of both subclones exhibited toxicity to larvae of Colorado potato beetle (Leptinotarsa decemlineata, a coleopteran insect). 73- and 65-kDa peptides that cross-reacted with an antiserum against the crystal protein of vat. tenebrionis were produced on expression in E. coli. Sporulating var. tenebrionis cells contain an immunoreactive 73-kDa peptide that corresponds to the expected product from the ORF of pNSBP544. However, isolated crystals primarily contain a 65-kDa component. When the crystal protein gene was shortened at the N-terminal region, the dominant protein product obtained was the 65-kDa peptide. A deletion derivative, p544Pst-Met5, was enzymatically derived from the 5.9 kb BamHI fragment upon removal of forty-six amino acid residues from the N-terminus. Expression of the N-terminal deletion derivative, p544Pst-Met5, resulted in the production of, almost exclusively, the 65 kDa protein. Recently, McPherson et al. (1988) Biotechnology 6:61-66 demonstrated that the Btt gene contains two functional translational initiation codons in the same reading frame leading to the production of both the full-length protein and an N-terminal truncated form.
Chimeric toxin genes from several strains of Bt have been expressed in plants. Four modified Bt2 genes from var. berliner 1715, under the control of the 2' promoter of the Agrobacterium TR-DNA, were transferred into tobacco plants (Vaeck et al. (1987) Nature 328:33-37). Insecticidal levels of toxin were produced when truncated genes were expressed in transgenic plants. However, the steady state mRNA levels in the transgenic plants were so low that they could not be reliably detected in Northern blot analysis and hence were quantified using ribonuclease protection experiments. Bt mRNA levels in plants producing the highest level of protein corresponded to .apprxeq.0.0001% of the poly(A).sup.+ mRNA.
In the report by Vaeck et al. (1987) supra, expression of chimeric genes containing the entire coding sequence of Bt2 were compared to those containing truncated Bt2 genes. Additionally, some T-DNA constructs included a chimeric NPTII gene as a marker selectable in plants, whereas other constructs carried translational fusions between fragments of Bt2 and the NPTII gene. Insecticidal levels of toxin were produced when truncated Bt2 genes or fusion constructs were expressed in transgenic plants. Greenhouse grown plants produced .apprxeq.0.02% of the total soluble protein as the toxin, or 3 .mu.g of toxin per g. fresh leaf tissue and, even at five-fold lower levels, showed 100% mortality in six-day feeding assays. However, no significant insecticidal activity could be obtained using the intact Bt2 coding sequence, despite the fact that the same promoter was used to direct its expression. Intact Bt2 protein and RNA yields in the transgenic plant leaves were 10-50 times lower than those for the truncated Bt2 polypeptides or fusion proteins.
Barton et al. (1987) Plant Physiol. 85:1103-1109 showed expression of a Bt protein in a system containing a 35S promoter, a viral (TMV) leader sequence, the Bt HD-1 4.5 kb gene (encoding a 645 amino acid protein followed by two proline residues) and a nopaline synthase (nos) poly(A)+ sequence. Under these conditions expression was observed for Bt mRNA at levels up to 47 pg/20 .mu.g RNA and 12 ng/mg plant protein, This amount of Bt protein in plant tissue produced 100% mortality in two days. This level of expression still represents a low level of mRNA (2.5.times.10.sup.-4 %) and protein (1.2.times.10.sup.-3 %).
Various hybrid proteins consisting of N-terminal fragments of increasing length of the Bt2 protein fused to NPTII were produced in E. coli by Hofte et al. (1988) FEBS Lett. 226:364-370. Fusion proteins containing the first 607 amino acids of Bt2 exhibited insect toxicity; fusion proteins not containing this minimum N-terminal fragment were nontoxic. Appearance of NPTII activity was not dependent upon the presence of insecticidal activity; however, the conformation of the Bt2 polypeptide appeared to exert an important influence on the enzymatic activity of the fused NPTII protein. This study did suggest that the global 3-D structure of the Bt2 polypeptide is disturbed in truncated polypeptides.
A number of researchers have attempted to express plant genes in yeast (Neill et al. (1987) Gene 55:303-317; Rothstein et al. (1987) Gene 55:353-356; Coraggio et al. (1986) EMBO J. 5:459-465) and E. coli (Fuzakawa et al. (1987) FEBS Lett. 224:125-127; Vies et al. (1986) EMBO J. 5:2439-2444; Gatenby et al. (1987) Eur. J. Biochem. 168:227-231). In the case of wheat .alpha.-gliadin (Neill et al. (1987) supra), .alpha.-amylase (Rothstein et al. (1987) supra) genes, and maize zein genes (Coraggio et al. (1986) supra) in yeast, low levels of expression have been reported. Neill et al. have suggested that the low levels of expression of .alpha.-gliadin in yeast may be due in part to codon usage bias, since .alpha.-gliadin codons for Phe, Leu, Ser, Gly, Tyr and especially Glu do not correlate well with the abundant yeast isoacceptor tRNAs. In E. coli however, soybean glycinin A2 (Fuzakawa et al. (1987) supra) and wheat RuBPC SSU (Vies et al. (1986) supra; Gatenby et al. (1987) Supra) are expressed adequately.
Not much is known about the makeup of tRNA populations in plants. Viotti et al. (1978) Biochim. Biophys. Acta 517:125-132 report that maize endosperm actively synthesizing zein, a storage protein rich in glutamine, leucine, and alanine, is characterized by higher levels of accepting activity for these three amino acids than are maize embryo tRNAs. This may indicate that the tRNA population of specific plant tissues may be adapted for optimum translation of highly expressed proteins such as zein. To our knowledge, no one has experimentally altered codon bias in highly expressed plant genes to determine possible effects of the protein translation in plants to check the effects on the level of expression.